Liquid flow photometer

ABSTRACT

A liquid flow photometer for fluorescence light and/or scattered light measurement, having a measuring area (37) where particles (38) flow through. The device comprises a first objective (3) focussing excitation light (5) in the measuring area (37) and also collecting fluorescence light emanating from the measuring area (37). The first objective (3) is modified by a slightly transparent central stop (39) placed in the rear focal plane of the objective. The device further comprises a second objective (23) collecting the light falling within the conic sector (40) obscured by the central stop (39). This light contains partly scattered light and excitation light. The corresponding photo-electric signal is divided in a pulsed (AC) component and a constant or slowly varying (DC) component. The DC component serves to correct one or both of the AC components representing the fluorescence and scattered light intensity in order to make them independent of variations in the excitation light intensity.

BACKGROUND OF THE INVENTION

The present invention relates to liquid flow photometers forfluorescence light and/or scattered light measurement, having ameasuring area where particles flow through.

Liquid flow photometry is a measuring technique increasingly more oftenapplied within several fields of cell biology, medicine andphysical-chemical measurements of particles of almost any material. In aliquid flow photometer photometric signals from single particles ofmicroscopic size are measured when they--carried by a laminar liquidflow--pass one by one through a focus of excitation light of highintensity. The particles are centered in the liquid flow by so calledhydrodynamic focussing. The types of photometric signals one is able toregister, are: (1) the particles' fluorescence, (2) the light scatteringcaused by the particles, and (3) light absorption caused by theparticles. The two first of these signal types have proved to beparticularly informative.

As a typical example of the application of liquid flow photometry onecan mention the measuring of the content of various components, as forinstance DNA or protein, in single cells. For this purpose the cells arestained with a fluorescent dye binding specifically and quantitativelyto the component in question. In this way every cell passing through thephotometer's excitation focus will cause a pulse of fluorescence light.This light is picked up by a light detector. The signal from this--whichis proportional to the cell's content of dye and thus of the cellcomponent in question--is measured and stored in an electronic memoryaccording to its size--a so called multichannel pulse height analyser.In this way several thousand cells can be measured per second with verygreat accuracy, and a histogram of the number of cells as a function ofthe cell's content of the cell component in question is obtained. Theexcitation light scattered by the cell as it passes the photometer'sfocus can be registered by another detector. Depending on the scatteringangle this signal gives information on the cell's cross section orvolume, and also on its inner structure and density.

In most types of liquid flow photometers the excitation light is inducedby a laser. The liquid flow carrying the particles passes through thelaser beam. The particles' fluorescence and light scattering areregistered by two separate optical systems located beside the laserbeam. In certain types of such liquid flow photometers the liquid flowis a free jet in the air. In others the liquid flow passes through aclosed chamber.

The types of liquid flow photometers mentioned above are complicatedlybuilt, and this makes them very expensive and difficult to operate. Anew type of liquid flow system, which can be applied as an accessory toa standard fluorescence microscope has been described in the NorwegianPatent Application No. 791229 Fitted with a suitable light detector themicroscope is converted to a liquid flow photometer which,--as regardsfluorescence detection--has proved to be up to the level of the far moreexpensive laser-based instruments, while it is also very easy tooperate. As described in the above mentioned patent application, in thisliquid flow system a hydrodynamically focussed flow of particles in alaminar liquid jet is induced by a nozzle. The nozzle is placed so thatthe liquid flow falls at an oblique angle onto a cover glass and is thusconverted to a flat, laminar flow moving along the surface of the coverglass. Because they are centered in the liquid jet, the particles willflow across the glass one by one within a narrow sector. The cover glassis placed so in relation to the microscope's objective, that theparticle flow on the glass passes through the objective's focus, i.e.the microscope's measuring area.

The above-described microscope has Epi-illumination, which means thatthe excitation light is focussed onto the measuring area by the sameobjective as that collecting the fluorescence light. The objective ispreferably of the oil immersion type to obtain maximum numericalaperture and correspondingly high sensitivity.

The liquid flow photometer described in the above-mentioned patentapplication can only measure the particles' fluorescence. Another of theinstrument's limitations is that the source of the excitation light is aconventional high pressure Hg- or Xe-lamp. The light intensity from suchlamps may vary somewhat, partly because the lamp's arc is not completelystable, and partly because the light emission from the lamp generallydecreases with the lamp's utility time. These variations reducereproducibility of the measurements.

SUMMARY OF THE INVENTION

It is therefore an object of the present invention to provide animproved microscope-based liquid flow photometer which makes it possibleto measure in combination with the particles' fluorescence lightintensity also the scattered light intensity in order to obtaininformation on the particles' size and structure.

It is a further object of the present invention to provide a liquid flowphotometer which makes it possible to measure the intensity of theexcitation light in the photometer's measuring area and to apply thissignal to correct the other photometric signals, i.e. fluorescence lightand scattered light for variations in this intensity in order to makethe instrument independent of variations in the intensity of theexcitation light.

In accomplishing the foregoing objects, there has been provided inaccordance with the present invention a liquid flow photometer forfluorescence light and/or scattered light measurement having a measuringarea where particles flow through. The photometer comprises:

(a) a first objective focussing excitation light in the measuring areaand also collecting fluorescence light emanating from the measuringarea, the objective being modified by a slightly transparent centralstop placed in the rear focal plane of the objective,

(b) a second objective collecting the light falling within the conicsector obscured by the central stop,

(c) a filter in the light path of the second objective absorbing thefluorescence light emanating from the measuring area,

(d) an adjustable aperture located in the image plane of the secondobjective limiting the image field of the second objective to the actualwidth of the measuring area,

(e) light detectors measuring the light intensity within the image planeof the first and second objective, and

(f) electronic amplifiers following the light detectors.

According to one preferred embodiment of the invention the secondobjective is placed so that its object plane falls in the measuring areaand that its aperture falls within the sector of the illumination fieldcaused by the central stop.

In accordance with another embodiment of the invention the measurementof scattered light intensity produced by the particles and excitationlight intensity in the measuring area is carried out by only one opticalsystem comprising the second objective, the filters, the adjustableaperture, one of the light detectors and one of the electronicamplifiers.

According to another preferred embodiment of the invention theelectronic amplifier following the light detector in connection with thesecond objective has separate outlets for the pulsed (AC) part and theconstant or slowly varying (DC) part of the signal from the lightdetector, the AC-signal corresponding to the scattered light intensityand the DC-signal corresponding the excitation light intensity.

According to another aspect of the invention, there has been provided aliquid flow photometer, comprising at least one pulse height compensatorcorrecting the AC-signals, corresponding to the fluorescence lightintensity and/or to the scattered light intensity by the DC-signalcorresponding to the excitation light intensity in such a way that theelectronic amplification is inversely proportional to the DC-signal.

Further objects, features and advantages of the present invention willbecome apparent to a person skilled in the art from the detaileddescription of a preferred embodiment which follows, when consideredtogether with the attached figures of drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a schematic setup of a complete liquid flow photometeraccording to the invention and

FIG. 2 shows in form of a block diagram the essential features formeasurements according to the invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

FIG. 1 shows an inverted microscope 1 with its Epi-illuminator 2 andfirst objective 3 below the stage 4. The illuminator receives its lightfrom a Hg- or Xe-lamp 5 via a collector-lens 6. The lamp 5 is fed by astabilized DC-power-supply 7. On the stage 4 is mounted the liquid flowsystem as described in the Norwegian patent application No. 791229. Themain part of this system is a nozzle 8 positioned under an oblique angleslightly above a cover glass 9. The nozzle 8 is connected to apressurized sheath fluid 10 and a disposable sample syringe 11 which ispressurized by a variable speed motor 12. The liquid jet leaving thenozzle 8 is converted to a flat, laminar flow moving along the surfaceof the cover glass 9. Because particles within the sample are centeredby hydrodynamic focussing in the liquid jet, they will flow across thecover glass 9 one by one within a narrow sector. The liquid flow will besucked off from the cover glass 9 by a drainage 13.

Visual observation of the particles and the particles fluorescence lightis possible through a binocular 14.

For photometric measurement of the fluorescence light the microscope 1is followed by a tube 15 which contains a measuring slit 16, adichromatic mirror 17, a mirror 18 and filters 19, 20. The filters,which transmit different parts of the spectrum, e.g. red and green, forthe purpose of spectral measurement, are followed by photomultipliers21, 22.

Above the stage 4 there is positioned a second objective 23 which has along focal distance. This objective is connected to a tube 24 whichcontains also a measuring slit 25, a filter 26 and a light detector 27.The electrical signal of this detector is amplified and devided in a DCand an AC part in an amplifier 28.

The photomultipliers 21, 22 are followed by amplifiers 29,30 and pulseheight compensators 31, 32, which are also connected to the DC outlet ofthe amplifier 28. The output signals of the pulse height compensators31, 32 are inversely proportional the DC signal of the amplifier 28##EQU1##

The pulse height compensator 32 can alternatively also be connected tothe AC outlet of the amplifier 28 so that this AC signal is corrected bythe DC signal in the same way as the AC signals from the amplifiers29,30.

The uncompensated scattered light AC signal and the compensated ACsignals (fluorescence and/or scattered light) are then fed to a separateunit for further pulse height analysis, computation, registration etc.FIG. 1 shows for example a dual parameter Multi-Channel-Pulse-HeightAnalyzer 33 indicating on a screen the number of particles (Z direction)having coinciding scattered and/or fluorescence light intensity (X-Ydirection).

FIG. 2 shows some more details of the essential features. TheEpi-illuminator 2 contains a further lens 34, a field diaphragm 35 and adichromatic mirror 36. This mirror reflects the excitation light via thefirst objective 3 into the measuring area 37 where the particles 38flow.

According to the invention the first objective 3 is modified by acentral stop 39 in the rear focal plane of the objective 3, This centralstop 39 obscures a conically formed sector 40 within the illuminationarea behind (over) the measuring area 37 of the microscope. Since theobjective 3 focusses the excitation light source 5 in the measuring area37 this results, in connection with the central stop 39, in a dark fieldcritical illumination. If the central stop 39 is perfectly opaque theconically formed sector 40 does not contain excitation light, but onlyfluorescence and scattered light from the particles 38 passing throughthe measuring area 37. The second objective 23 is placed behind (above)the measuring area 37 in such a way that its entire aperture fallswithin the conic, dark sector 40 and thus only catches fluorescence andscattered light. The aperture of the objective 23 can be made tocorrespond to the sector 40 by an adjustable diaphragm 41 located infront of the objective 23. The measuring slit 25 located in the imageplane of the objective 23 limits the light detected by the detector 27to that part of the measuring area where the fluorescence is measured.

The fluorescence light is eliminated by using a suitable light filter 26in the light path of the second objective 23. Thus the light falling onthe light detector 27 placed behind this filter, will only be scatteredlight from the particles 38.

According to the present invention the above mentioned central stop 39is not fully opaque but slightly transparent, that means it has a veryfaint light transmission.

In this way the above mentioned dark, conic sector 40 will contain avery faint component of direct excitation light also falling on thelight detector 27.

The light scattered by the particles 38 appears as short pulses, whilethe direct excitation light only varies slowly with time. Thus thesignal from the light detector 27 behind the second objective 23 willcontain two components:

(a) an AC component in the form of short pulses representing thescattered light intensity and

(b) a DC component representing the intensity of the direct excitationlight.

Both signals are separated and amplified in the amplifier 28.

The fluorescence light emanating from the particles 38 in the measuringarea 37 is collected by the first objective 3 and passes the dichromaticmirror 36. The measuring slit 16 limits the field of the measuring area37 and the light filters 19 and 20 serve for spectral measurements ofthe fluorescence light. Because of the dichromatic mirror 36 the signalfrom the light detector 22 following the first objective 3 will containonly an AC component in form of short pulses representing thefluorescence light intensity. These pulses are amplified in theamplifier 30.

The signal from the light detector 27 and the amplifier 28 representingthe above mentioned DC component is fed as a correction signal to thepulse height compensators for the fluorescence light intensity 31 andthe scattered light intensity 32. These corrections are made so that theamplification level is inversely proportional to the DC signal ##EQU2##As the pulses entering the pulse height compensators are proportional tothe intensity of excitation light and thus to the above mentioned DCcomponent, the pulses coming out of the compensators will be independentof this light intensity which is one object of the present invention.

What is claimed is:
 1. A liquid flow photometer for fluorescence lightand/or scattered light measurement, having a measuring area (37) whereparticles (38) flow through comprising:(a) a fist objective (3)positioned for focussing excitation light (5) in the measuring area (37)and also for collecting fluorescence light emanating from said measuringarea, said objective being modified by a slightly transparent centralstop (39) placed in the rear focal plane of said objective for causingonly a very faint light transmission in a sector of said excitationlight, (b) a second objective (23) positioned for collecting the lightfalling within said sector (40) caused by said central stop (39), (c) afilter (26) in the light path of said second objective (23) absorbingthe fluorescence light emanating from said measuring area (37), (d) anadjustable aperture (25) located in the image plane of said secondobjective (23) limiting the image field of said second objective (23) tothe actual width of said measuring area (37), (e) light detectorscomprising a first light detector positioned for measuring the lightintensity collected by said first objective and a second light detectorpositioned for measuring the light intensity collected by said secondobjective, and (f) electronic amplifiers following said light detectors.2. A liquid flow photometer as defined in claim 1 wherein said secondobjective (23) is placed so that its object plane falls in saidmeasuring area (37) and that its aperture falls within said sector ofthe illumination field (40) caused by said central stop (39).
 3. Aliquid flow photometer as defined in claim 1 wherein the measurement ofscattered light intensity produced by said particles (38) and excitationlight intensity in the measuring area (37) is carried out by a commonoptical system comprising said second objective (23), said filter (26),said adjustable aperture (25), one of said light detectors (27) and oneof said electronic amplifiers (28).
 4. A liquid flow photometer asdefined in claim 3 wherein said electronic amplifier (28) following saidlight detector (27) in connection with said second objective (23) hasseparate outlets for the pulsed (AC) part and the constant or slowlyvarying (DC) part of the signal from said light detector (27), saidAC-signal corresponding to the scattered light intensity and saidDC-signal corresponding to the excitation light intensity.
 5. A liquidflow photometer as defined in claim 4 comprising at least one pulseheight compensator (31, 32) correcting the AC-signals, corresponding tothe fluorescence light intensity and/or to the scattered light intensityby the DC-signal corresponding to the excitation light intensity in sucha way that the electronic amplification is inversely proportional tosaid DC-signal.
 6. A liquid flow photometer as defined in claim 1further including an adjustable diaphragm positioned to match the sizeof the aperture of said second objective to said sector caused by saidslightly transparent central stop.